KR101642939B1

KR101642939B1 - Iron Oxide Nano Particle Capsulated Polymer Nano Capsule, Fabrication Method of Polymer Nano Capsule and the MRI Contrast Agents Using Thereof

Info

Publication number: KR101642939B1
Application number: KR1020100084756A
Authority: KR
Inventors: 권은별; 전봉식; 김응규; 박주영; 명완재
Original assignee: 한화케미칼 주식회사
Priority date: 2010-08-31
Filing date: 2010-08-31
Publication date: 2016-07-26
Also published as: EP2621852A4; WO2012030166A2; JP2013541509A; WO2012030166A3; US9028875B2; KR20120020861A; CN103201211B; JP5685647B2; EP2621852A2; US20140328768A1; CN103201211A

Abstract

The present invention relates to an iron oxide nanocapsule which is excellent in water-dispersibility, is safe to the living body and has excellent MRI contrast, a method for producing the same, and a contrast agent using the same. More particularly, The iron oxide nanocapsules are produced by preparing iron oxide nanoparticles having a hydrophobic ligand bound thereto by pyrolyzing the complex and encapsulating the iron oxide nanoparticles in a capsule material of a carboxymethyldextran-dodecylamine conjugate or a dextran-linoleic acid conjugate .

Description

TECHNICAL FIELD [0001] The present invention relates to an iron oxide nanocapsule, a method for producing the same, and a magnetic resonance imaging contrast agent containing the same. [0002] Iron oxide nanoparticles,

The present invention relates to an iron oxide nanocapsule excellent in water-dispersibility, safe for living body, excellent in MRI contrast, a method for producing the same, and a contrast agent using the same.

Nanomaterials are materials with sizes ranging from 1 nm to 100 nm, and can be synthesized in various forms such as spheres, plates, and tubes. Utilizing the unique properties of nanomaterials, which are different from the previous bulk materials, active application technologies are being developed in various fields such as electronics, information, environment, energy, and medicine.

In particular, magnetic nanoparticles having magnetic properties in nanomaterials are being studied over a wide range of applications including separation of biomaterials, contrast imaging agents for MRI, biosensors, drug / gene delivery, and magnetic high temperature treatment. Magnetic resonance imaging Resovist (Bayer Schering), an intravenous contrast agent based on iron oxide nanoparticles, is currently in clinical use and Combidex (AMAG), a lymphatic contrast agent, is known to be in clinical trials.

However, the iron oxide nanoparticles, which are magnetic nanoparticles used in this commercial contrast agent, were synthesized by coprecipitation using a metal salt in aqueous solution. Therefore, it is difficult to adjust the size and the monodispersity is not good. In addition, since it is synthesized at room temperature, there is a disadvantage that the crystallinity of the nanoparticles is low.

Another method for preparing iron oxide nanoparticles is the synthesis using pyrolysis. The pyrolysis method uses an organic solvent, a metal precursor and an organic surface stabilizer to improve the stability of the particles, and aging the mixture by heating to the boiling point of the solvent. Therefore, the reaction temperature can be easily controlled and boiling points By using a solvent, nanoparticles of various sizes can be synthesized. Such synthesized nanoparticles have superior monodispersity and crystallinity to nanoparticles synthesized in aqueous solution, and thus exhibit excellent physical properties in application fields.

However, since the pyrolysis method synthesizes nanoparticles in an organic solvent, the surface of the synthesized iron oxide nanoparticles is surrounded by an organic material, and this is not suitable for application to nano-bio fields and the like.

Therefore, additional nanoparticle surface modification is needed as a stable and water-dispersible material in vivo. There are various methods of surface modification of nanoparticles. Typical examples are a ligand exchange method for converting organic substances on the surface of nanoparticles into hydrophilic materials and an encapsulation method for encapsulating the surface with a hydrophilic material while leaving the organic material intact. Encapsulation methods include encapsulation of one particle, Is encapsulated.

In the case of the encapsulation process, when an amphipathic substance having both a hydrophilic and a hydrophobic substance is used, the hydrophobic region of the amphipathic compound binds to the surface of the nanoparticle, and the hydrophilic region of the amphipathic compound is distributed in the outermost portion of the capsule nanoparticle Insoluble nanoparticles can be stably dispersed in a water-soluble solvent, so that the bioavailability can be maximized.

The present invention provides an iron oxide nanocapsule having extremely excellent dispersibility in a water-soluble solvent, excellent biostability, and excellent MRI contrast performance, and a method for producing the same.

The manufacturing method of the present invention will be described in detail with reference to the accompanying drawings. The following drawings are provided by way of example so that those skilled in the art can fully understand the spirit of the present invention. Therefore, the present invention is not limited to the following drawings, but may be embodied in other forms, and the following drawings may be exaggerated in order to clarify the spirit of the present invention. Also, throughout the specification, like reference numerals designate like elements.

Hereinafter, the technical and scientific terms used herein will be understood by those skilled in the art without departing from the scope of the present invention. Descriptions of known functions and configurations that may be unnecessarily blurred are omitted.

The method for preparing iron oxide nanocapsules according to the present invention comprises pyrolyzing an iron complex to prepare iron oxide nanoparticles to which a hydrophobic ligand is bound and preparing the iron oxide nanoparticle by dissolving it in a carboxymethyldextran-dodecylamine conjugate or a dextran-linoleic acid conjugate Encapsulated in capsule material to produce iron oxide nanocapsules.

In detail, the process for producing iron oxide nanocapsules according to the present invention comprises the steps of: a) preparing a carboxymethyldextran-dodecylamine conjugate of the following formula 1 or a capsule material of a dextran-linoleic acid conjugate of the following formula 2 Producing; b) pyrolyzing an iron complex in which iron is a central atom and an organic acid group including C4 to C25 is bonded with a ligand to produce iron oxide nanoparticles having a hydrophobic ligand bound thereto; c) dissolving the capsule material in a buffer solution to prepare a capsule aqueous solution, and dispersing the iron oxide nanoparticles in a nonpolar organic solvent to prepare a nanoparticle dispersion; d) dropping the nanoparticle dispersion in the capsule aqueous solution and stirring the mixture to prepare an iron oxide nanocapsule in which a plurality of iron oxide nanoparticles are encapsulated in a capsule material; And e) removing the non-polar organic solvent contained in the iron oxide nanocapsule dispersion of step d) using volatilization to prepare an iron oxide nanocapsule water dispersion.

(Formula 1)

(2)

Wherein R is H or CH ₂ COOH, and n is an integer of 1 to 5000, wherein x is an integer of 1 to 1000, and y is an integer of 1 to 1000.

The carboxymethyldextran-dodecylamine conjugate of Formula 1 or the capsule material of dextran-linoleic acid conjugate of Formula 2 forms micelles in a water solvent and is synthesized by pyrolysis to form hydrophobic ligands And the encapsulated iron oxide nanoparticles have an extremely excellent dispersibility with respect to a water-soluble solvent.

Further, the iron oxide nanoparticles prepared by thermally decomposing an iron salt having an organic acid group bonded to the iron center can be precisely controlled in size from several to several tens nanometers according to the thermal decomposition temperature and time, and the iron oxide nanoparticles having a very uniform size It is a feature of nanoparticles.

Furthermore, dextran is a water-soluble polysaccharide composed of glucose, which is one of D-glucose. Dextrans is degraded by lactic acid bacteria belonging to Royokonostock to polymerize D-glucose. It has been approved by the FDA and used as a plasma expanding agent. Especially, it has been used as a coating material of iron oxide nanoparticles of Feridex (AMAG), a commercial contrast agent. Carboxymethyldextran is a dextran derivative having a carboxy group attached to dextran. It is known that 1.1 to 1.5 mmol of carboxy group per g of tran is attached. It has been used as an iron oxide nanoparticle coating material for Resovist, a commercial contrast agent based on iron oxide nanoparticles, and as an iron oxide nanoparticle coating material for combidex, a lymphocyte contrast agent currently in clinical trials. As a result, iron oxide nanoparticles which are very stable in the living body while having a very high aqueous dispersion are produced, and encapsulated with a carboxymethyldextran-dodecylamine conjugate or a dextran-linoleic acid conjugate of the above formula (2) Iron oxide nanocapsules of extremely uniform size are produced, and capillary blood vessels are blocked during injection or absorption / diffusion to tissues other than the target tissue is prevented.

Specifically, in the first embodiment of the present invention wherein the capsule material is a carboxymethyldextran-dodecylamine conjugate, the capsule material is a carboxymethyldextran-dodecylamine conjugate, A1-1) mixing a solution of carboxymethyldextran and a solution of dodecylamine; a1-2) adding EDC (1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide) and NHS (n-hydroxysuccinicimide); And a3) dialysis and lyophilization.

The average molecular weight of the carboxymethyldextran is 500 to 1,000,000 Da, preferably 10,000 to 100,000.

The carboxymethyldextran solution is prepared by adding 10 to 15 times (by weight) DMSO (by weight) based on the weight of water of the carboxymethyldextran aqueous solution to an aqueous solution of carboxymethyldextran mixed with carboxymethyldextran: water in a ratio of 1: 3 to 5 and the dodecylamine solution is preferably prepared by mixing dodecylamine: dimethyl sulfoxide (DMSO) in a ratio of 1: 8 to 12: 25 to 35 by weight.

When the two solutions are mixed in the step a1-1), the carboxymethyldextran solution and the dodecylamine solution are mixed so that the dodecylamine: carboxymethyldextran ratio is 1: 1 to 10: 1. The degree of hydrophilicity and hydrophobicity of the conjugate produced by the mixing conditions of the carboxymethyldextran solution and the dodecylamine solution can be controlled.

In the step a1-2), it is preferable that the EDC and the NHS are added such that the dodecylamine: EDC: NHS is 1: 0.3 to 0.7: 0.1 to 0.4.

Specifically, in the second aspect of the present invention wherein the capsule material is a dextran-linoleic acid conjugate, the capsule material comprises a2-1) mixing a dextran solution and a linoleic acid solution; ^{a2-2) DCC (n, n '} -dicyclohexylcarbodiimide) , and adding a DMAP (dimethylaminopyridine); a3) dialysis and lyophilization.

The average molecular weight of the dextran is 100 to 100,000 Da, preferably 5,000 to 10,000.

The dextran solution is preferably prepared by mixing dimethylsulfoxide (DMSO) at a weight ratio of 1:50 to 80, and the linoleic acid solution is prepared by mixing linolenic acid: dimethyl sulfoxide (DMSO) at a weight ratio of 1: 8 to 14 Is preferably produced.

In the mixing of step a2-1), the dextran solution and the linoleic acid solution are preferably mixed so that dextran: linoleic acid is in a weight ratio of 1: 2 to 5. The degree of hydrophilicity and hydrophobicity of the conjugate produced by the mixing conditions of the dextran solution and the resolvolenic acid solution can be controlled.

Preferably, the DCC and DMAP are added so that dextran: DCC: DMAP is 1: 1.5 to 2: 0.3 to 0.8 in the step a2-2). At this time, the DCC and DMAP are preferably added in dissolved form in DMSO.

In the method for producing iron oxide nanocapsules (including the first aspect and the second aspect) according to the present invention, the buffer solution in step c) is a phosphate buffer solution, and the nonpolar organic solvent is chloroform.

Preferably, the capsule aqueous solution is prepared by mixing the capsule material: buffer solution at a weight ratio of 1: 50 to 500, and the nanoparticle dispersion is prepared by mixing iron oxide nanoparticles: nonpolar organic solvent at a weight ratio of 1:10 to 100 .

The amount of iron oxide nanoparticles enclosed in a single capsule, the average size and capsule distribution of the capsules, and the degree of coagulation of the iron oxide nanoparticles to be encapsulated are controlled by the size of the iron oxide nanoparticles and the detailed execution conditions of step d).

The average size (diameter) of the iron oxide nanoparticles is preferably in the range of 3 nm to 20 nm in order to maximize the MRI contrast performance with magnetic properties suitable for the contrast agent. As described above, the iron oxide nanoparticles are iron oxide nanoparticles in which iron is a central atom and hydrophobic ligands are combined with pyrolysis of an iron complex having an organic acid group including C4 to C25 bound thereto as a ligand. The iron complex includes an iron oleate complex, and the iron oxide nanoparticles to which the hydrophobic ligand is bound include iron oxide nanoparticles bound with an oleate.

In detail, the iron oxide nanoparticles are preferably prepared by reacting the above-described iron complex and the fatty acid containing oleic acid at 300 to 350 DEG C for 20 to 90 minutes. For a more detailed method of producing iron oxide nanoparticles, see PCT / KR2005 / 004009.

The iron oxide nanoparticle dispersion is dispersed so that the weight ratio of the capsule material: iron oxide nanoparticles is 1: 0.05 to 0.25 in the step (d) in order to reduce the variation in the amount of encapsulation of the iron oxide nanoparticles per capsule and to effectively encapsulate the iron oxide nanoparticles. It is preferable to drop it in an aqueous solution of the capsule.

It is preferable that the capsule is controlled at an average size of 100 to 500 nm and the capsule is prepared at a rate of 0.1 to 3 ml / min as in step d) in order to prepare a capsule having an extremely uniform size, Is preferably performed at 20,000 to 30,000 rpm. At this time, it is preferable that the iron oxide nanoparticle dispersion is dropped into the capsule aqueous solution and the stirring is performed.

According to the manufacturing method of the present invention, a plurality of iron oxide nanoparticles having an average size of 5 nm to 20 nm and having a hydrophobic ligand bound thereto are encapsulated in a capsule material that is a carboxymethyldextran-dodecylamine conjugate or a dextran-linoleic acid conjugate Iron oxide nanocapsules are prepared, and iron oxide nanocapsules having an average size of 100 nm to 500 nm and having extremely uniform dispersion properties in a very uniform manner are prepared.

The MRI contrast agent containing the iron oxide nanocapsules is particularly suitable as an hepatic contrast agent, and the iron oxide nanocapsule has excellent liver tissue selectivity and excellent contrast enhancement.

The method for preparing an iron oxide nanocapsule according to the present invention is characterized in that a plurality of iron oxide nanoparticles are encapsulated in a single capsule to have a very excellent image contrast of a magnetic resonance image due to the agglomeration effect between the iron oxide nanoparticles, It is possible to prevent the capsules from being absorbed and distributed to tissues other than the tissue to be contrasted (particularly, liver tissue), to have a very excellent dispersibility with respect to the aqueous solution, and to have a stable effect on the living body.

1 is a TEM (Transmission Electron Microscope) photograph of the iron oxide nanoparticles prepared in Preparation Example 1,
2 is a TEM photograph of the iron oxide nanoparticles prepared in Preparation Example 2,
3 is a TEM photograph of the iron oxide nanoparticles prepared in Production Example 3,
4 is a FT-IR analysis result of the carboxymethyldextran-dodecylamine amphipathic polymer complex prepared in Example 1,
FIG. 5 is a TEM photograph of an iron oxide capsule encapsulating iron oxide nanoparticles to which olate was attached to the carboxymethyldextran-dodecylamine conjugate capsule material prepared in Example 2,
6 is a TEM photograph of the iron oxide capsule prepared in Example 3,
7 is a TEM photograph of the iron oxide capsule prepared in Example 4,
8 is a graph showing the R2 relaxivity of an iron oxide capsule encapsulated with 10 nm iron oxide nanoparticles,
FIG. 9 is a graph showing the R2 relaxivity of an iron oxide capsule encapsulated with 11 nm iron oxide nanoparticles,
10 is a graph showing the results of measuring R2 relaxivity of iron oxide encapsulated 15 nm iron oxide nanoparticles,
11 is a view showing an in-vivo MRI using the iron oxide capsule according to the present invention,
FIG. 12 is a graph showing relaxivity by digitizing a signal value of an image obtained by measurement of liver magnetic resonance imaging using an iron oxide capsule according to the present invention,
13 is a TEM photograph of the carboxymethyldextran-iron oxide nanoparticles prepared in Comparative Example 1,
14 is a TEM photograph of the carboxymethyldextran-iron oxide nanoparticles prepared in Comparative Example 2,
15 is a FT-IR analysis result of the dextran-linoleic acid conjugate prepared in Example 7,
16 is a TEM photograph of the capsule particles prepared in Example 8,
17 is a view showing an in-vivo MRI using the iron oxide capsule according to the present invention,
FIG. 18 is a graph showing relaxivity by quantifying the signal value of an image obtained by measurement of intra-liver magnetic resonance imaging using the iron oxide capsule according to the present invention,
19 is a TEM photograph of the dextran-iron oxide nanoparticles prepared in Comparative Example 3. Fig.

Hereinafter, the present invention will be described in more detail with reference to the following examples and comparative examples, but the present invention is not limited to these examples.

[Preparation Example 1] Synthesis of 10 nm iron oxide nanoparticles

10.8 g of iron chloride (FeCl ₃ .H ₂ O, 40 mmol) and 36.5 g of sodium oleate (120 mmol) were dissolved in a mixed solvent containing 80 ml of ethanol, 60 ml of distilled water and 140 ml of hexane and the mixture was heated to 57 ° C And maintained at the same temperature for 1 hour.

During this process, the initial vermilion clears in the aqueous phase, and the initial transparent organic phase is reddish, indicating that the oleic acid complex has been successfully synthesized.

Upon completion of the reaction, the upper organic layer containing the iron oleate complex was separated, and then the hexane was evaporated, resulting in a viscous liquid form.

36 g of the iron oleate complex thus prepared were added to a mixture of 200 g of octadecene and 5.7 g of oleic acid. The resultant mixture was heated from room temperature to a temperature of 70 ° C at a rate of 2.5 ° C / min under vacuum, and then maintained at the same temperature for 1 hour to remove residual solvent and moisture from the reaction product.

Thereafter, the mixture was heated to 320 DEG C at a rate of 2.5 DEG C / min in a nitrogen atmosphere, and aged while being kept at the same temperature for 30 minutes. During this process, a violent reaction occurred, , Indicating that the oleic acid iron complex was completely decomposed and iron oxide nanoparticles were produced.

After the reaction was completed, air was injected and oxidized when the temperature was below the auto-ignition temperature (150 ° C.) during the natural cooling process. The resultant nanoparticle-containing solution was cooled to room temperature and black precipitate was formed by adding a hexane-acetone mixture solution having a volume ratio of 1: 5 in an amount corresponding to three times the volume of the mother liquor, followed by centrifugation (rpm = 2,000). The resulting supernatant was discarded.

The hexane and acetone mixed solution was added and centrifuged at least twice. The hexane and acetone contained in the residue were removed by drying to obtain an iron oxide nanoparticle having an oleate-re-dispersed easily in hexane .

FIG. 1 shows TEM (Transmission Electron Microscope) images of the prepared iron oxide nanoparticles. As a result of TEM observation, it was confirmed that an oxide-coated iron oxide nanoparticles having an average diameter of 10 nm were produced. It was confirmed that iron oxide nanoparticles having a very uniform size were produced.

[Preparation Example 2] Synthesis of 11 nm iron oxide nanoparticles

Iron oxide nanoparticles were synthesized using the same reaction conditions as described in Preparation Example 1, except that the aging time was 1 hour during the synthesis.

FIG. 2 shows TEM (Transmission Electron Microscope) images of the prepared iron oxide nanoparticles. It is confirmed that spherical iron oxide nanoparticles having an average diameter of 11 nm are attached to the oleate, and the iron oxide nanoparticles having a very uniform particle size Was produced.

[Preparation Example 3] Synthesis of 15 nm iron oxide nanoparticles

Iron oxide nanoparticles were synthesized using the same reaction conditions as described in Preparation Example 1, except that 1-eicosene was used instead of octadecene during the synthesis and the reaction was aged at 330 ° C for 1 hour.

FIG. 3 shows TEM (Transmission Electron Microscope) images of the iron oxide nanoparticles produced. It was confirmed that spherical iron oxide nanoparticles having an average particle diameter of 15 nm were attached to the oleate, and the spherical iron oxide nanoparticles Was produced.

[Example 1] Synthesis of carboxymethyldextran-dodecylamine conjugate

To synthesize a carboxymethyldextran-dodecylamine conjugate, 5 g of carboxymethyldextran (average molecular weight 14,000) was completely dissolved in 20 ml of distilled water and mixed with 250 ml of DMSO (dimethylsulfoxide) to prepare a carboxymethyldextran solution . 1.4 g (7.5 mmol) of dodecylamine, 10 ml of CHCl ₃ and 40 ml of DMSO were mixed to prepare a dodecylamine solution.

After mixing two solutions at a dropping rate of 1 ml / min by dropping a dodecylamine solution into the prepared carboxymethyldextran solution, 0.7 g of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide And 0.35 g of NHS (n-hydroxysuccinicimide) were dissolved in 30 ml of DMSO and 10 ml of NHS solution, respectively.

Thereafter, the mixture was stirred at room temperature for 24 hours, filtered using a filter paper, and then excess ethanol was added to form a white precipitate. The precipitate was separated by centrifugation (rpm = 3,000).

The precipitate was dispersed in distilled water, dialyzed in distilled water for 3 days, and lyophilized to obtain a white solid state carboxymethyldextran-dodecylamine conjugate.

4 shows FT-IR analysis results of carboxymethyldextran-dodecylamine amphipathic polymer complex. A new peak at 1656 cm ^-1 and 1593 cm ^-1 was observed, which means C = O stretching (amide Ⅰ) and NH deformation (amide Ⅱ) in the amide bond, respectively. In addition, CH stretching peaks were observed at 2933 cm ^-1 and 2875 cm ^-1 . Based on the above results, it was found that the carboxymethyldextran-dodecylamine conjugate was successfully synthesized.

[Example 2] Encapsulation of iron oxide nanoparticles using carboxymethyldextran-dodecylamine conjugate

55 mg of the carboxymethyldextran-dodecylamine conjugate prepared in Example 1 and 25 ml of PBS were mixed and sonicated for 10 minutes to prepare a capsule aqueous solution. The aqueous solution of the iron oxide nano-particles having an average particle diameter of 10 nm prepared in Preparation Example 1 11 mg of the particles were dispersed in 0.5 ml of chloroform and ultrasonicated for 10 minutes to prepare a nanoparticle dispersion.

The nanoparticle dispersion was dropped into a carboxymethyldextran-dodecylamine conjugate solution (capsule aqueous solution) dropwise at a rate of 1 ml / min for 10 minutes using a homogenizer (rpm = 26,000) . The solution in which the capsule particles were formed was removed by using a vacuum solvent evaporator to remove chloroform contained in the solution.

FIG. 5 is a TEM photograph showing the negative staining of the iron oxide capsule encapsulated with the iron oxide nanoparticles to which olate is attached to the carboxymethyldextran-dodecylamine conjugate capsule material prepared through the above process. TEM observation revealed that the iron oxide nanoparticles were encapsulated, and it was confirmed that a plurality of iron oxide nanoparticles were encapsulated in a single capsule. The size of the capsules was measured by dynamic light scattering (DLS, Melvern Zetasizer), and the average particle size (Z-average) was 150 nm.

[Example 3] Encapsulation of iron oxide nanoparticles using carboxymethyldextran-dodecylamine conjugate

Capsule particles were prepared in the same manner as described in Example 3, except that iron oxide nanoparticles having an average particle diameter of 11 nm prepared in Preparation Example 2 were used. FIG. 6 is a TEM photograph of the prepared capsules, and the average particle sizes of the prepared capsules were 144 nm, respectively.

[Example 4] Encapsulation of iron oxide nanoparticles using carboxymethyldextran-dodecylamine conjugate

Capsule particles were prepared in the same manner as described in Example 3 except that iron oxide nanoparticles having an average particle diameter of 15 nm prepared in Preparation Example 3 were used. FIG. 7 is a TEM photograph of the prepared capsule, and the average particle diameter of the prepared capsule was 188 nm.

[Example 5] Measurement of relaxation performance of an iron oxide capsule containing iron oxide nanoparticles encapsulated in a carboxymethyldextran-dodecylamine capsule material

The nanocapsules prepared in Examples 2, 3 and 4 were each dispersed in PBS to prepare solutions having concentrations of 0.36 μg / ml, 0.19 μg / ml and 0.12 μg / ml, and diluted with PBS (Bospor 47/40, Bruker Biospin MRI GmbH) were used to measure the extracorporeal magnetic resonance relaxation performance. The T2 relaxation time measurement was performed using MSME (Multi Slice Multi-Echo sequence) pulse sequence, and specific parameters are shown in Table 1.

division parameter Remarks TR (Reopetition time) 10,000 ms TE (Echo time) 8 to 2048 ms 256 times at 8 ms intervals FOV 60 X 40 mm Resolution 0.234 X 0.156 mm / pixel

FIG. 8 is a graph of R2 relaxivity measurement of capsules using 10 nm iron oxide nanoparticles, and R2 relaxivity value is 298 mM ^-1 s ^-1 . FIG. 9 is a graph showing R2 relaxivity measurement results of capsules using 11 nm iron oxide nanoparticles, and R2 relaxivity value is 316 mM ^-1 s ^-1 . FIG. 10 is a graph of R2 relaxivity measurement of capsules using 15 nm iron oxide nanoparticles, and R2 relaxivity value is 340 mM ^-1 s ^-1 .

[Example 6] Measurement of internal magnetic resonance relaxation performance of iron oxide capsule encapsulated with iron oxide nanoparticles in a carboxymethyldextran-dodecylamine capsule material

Magnetic Resonance Imaging of Iron Oxide Nanoparticles Encapsulated in Carboxymethyldextran-Dodecylamine Amphipathic Polymer Capsules To measure the liver contrast agent performance, a BGA12 gradient was measured in a 4.7 T magnetic resonance imaging device (Biospec 47/40, Bruker Biospin MRI GmbH) The in vivo T2 relaxation performance was measured using a coil.

The mice used in the experiment were 5-week-old male Balb / c mice and the mice weighed about 20-25 g. The mice were anesthetized and placed horizontally in the MRI apparatus to observe the coronal plane. During the whole experimental period, the mice were kept in motion with little breathing to examine the liver tissue on the same side.

The iron concentration of the iron oxide capsule was analyzed by ICP-AES, and 200 μl of the solution was injected through the tail vein of the rat at one time. The concentration of the final solution was 1 mg Fe / kg. < / RTI > T2 relaxation time measurement was performed using RARE (Rapid Acquisition with Refocused Echoes) pulse sequence, and specific parameters are shown in Table 2.

division parameter Remarks TR (Reopetition time) 3,500 ms TE (Echo time) 36 ms 256 times at 8 ms intervals FOV 60 X 40 mm Resolution 0.234 X 0.156 mm / pixel Slice thickness 1 mm

To quantitatively evaluate the T2 attenuation effect of iron oxide capsules encapsulating iron oxide nanoparticles in a carboxymethyldextran-dodecylamine capsule material, one section of the liver tissue was selected and the whole liver portion was selected as ROI (Region of Interests) Signal Intensity (SI) was analyzed. In order to maximize the reliability of the obtained signal intensity, Agarose 1 wt% solution was placed in a 200 μl tube, cooled, solidified, fixed in the vicinity of the abdominal cavity of rats, and used as a control.

FIG. 11 shows a magnetic resonance imaging image when the iron oxide capsule prepared in Example 2 is used as a contrast agent. When the images before contrast injection and after contrast injection are compared, it can be seen that the liver color of the mouse has changed to black It was found that an iron oxide capsule encapsulating iron oxide nanoparticles in the carboxymethyldextran-dodecylamine amphipathic polymer capsule material could be used as an hepatic contrast agent.

FIG. 12 is a graph showing relaxivity by quantifying the signal value of an image obtained by measurement of intracoronary magnetic resonance imaging using the iron oxide capsule prepared in Example 2 as a contrast agent. The ΔR2 value was measured to be 47-60%.

[Comparative Example 1] Encapsulation of iron oxide nanoparticles using carboxymethyldextran

120 mg of iron oxide nanoparticles having an average particle diameter of 10 nm were dispersed in 20 ml of hexane, and the mixture was stirred in the reactor. 20 mg of carboxymethyldextran was dissolved in 20 ml of distilled water and dropped onto the iron oxide nanoparticle solution by a dropwise method. The mixture was stirred at 50 ° C for 1 hour. The iron oxide nanoparticles in the hexane layer were hydrophilized by carboxymethyldextran, The hexane layer was cleared, and the water layer showed brownish color, which is the color of the iron oxide nanoparticles. Distilled water and ethanol were added to the mixture to form a precipitate using a centrifugal separator, and the formation of encapsulated particles was observed through TEM.

13 is a TEM photograph of the carboxymethyldextran-iron oxide nanoparticles prepared by the above method. It is not encapsulated, and the iron oxide nanoparticles are formed into a large lump and clump together.

From the above results, it was found that capsule nanoparticles could not be formed when carboxymethyldextran, which is a hydrophilic substance, was used alone without using an amphipathic molecule.

[Comparative Example 2] After hydrophilization using a surfactant, carboxymethyldextran was encapsulated with iron oxide nanoparticles

3 g of sodium dodecyl sulfate (SDS) was dissolved in 18 ml of distilled water and mixed with a solution of 0.03 g of iron oxide nanoparticles dispersed in 3 ml of chloroform. The chloroform was evaporated using a vacuum solvent evaporator to obtain a solution in which the iron oxide nanoparticles coated with the surfactant were dispersed. 0.01 g of carboxymethyldextran was dissolved in 5 ml of distilled water, and the solution was dropped. After stirring at 50 ° C for 1 hour, distilled water and ethanol were added and a precipitate was formed using a centrifuge. In order to observe the polymer part well, negative staining was performed and the shape of the particles was observed through TEM.

FIG. 14 is a TEM photograph of the carboxymethyldextran-iron oxide nanoparticles produced as a result of the above. It was found that the exposed portion was carboxymethyldextran and the iron oxide nanoparticles were encapsulated. However, when the encapsulation is performed after hydrophilization using a surfactant without using an amphipathic molecule, there are problems in that the size of the particles is not easily controlled and dispersion stability is poor.

[Example 7] Synthesis of dextran-linoleic acid amphipathic conjugate

To synthesize dextran-linoleic acid conjugate, 0.8 g of dextran (average molecular weight of 10,000) was dissolved in 50 ml of DMSO to prepare a dextran solution. 2.212 ml (7.0 mmol) of linoleic acid was dissolved in 20 ml of DMSO to prepare a linolenic acid solution Respectively. It was added dropwise to a solution of linoleic acid in the manufacture dextran solution (dropping rate 1 ml / min) and then a solution of the two ^{solutions, DCC (n, n '-dicyclohexylcarbodiimide} ) 1.12 ml (7.0 mmol), DMAP (dimethylaminopyridine) 0.44 g (3.5 mmol) were dissolved in 20 ml of DMSO, and the mixture was reacted at 25 ° C for 24 hours.

After filtration using filter paper, DCU (Dicyclohexylurea) as a by-product was filtered out, and methanol / acetonitrile mixed at a ratio of 1: 2 was added to the filtered solution to yield a pale yellow precipitate. After filtering the solution, the precipitate remaining on the filter paper was washed three times with a mixed solution of methanol and acetonitrile 1: 2 and dried. The dried white solid material was dissolved in distilled water and dialyzed and lyophilized to obtain a solid dextran-linoleic acid conjugate.

15 is a FT-IR analysis result of a dextran-linoleic acid conjugate. The peak of the ester group was confirmed at 1745 cm ^-1 , indicating that the dextran-linoleic acid conjugate was successfully synthesized.

[Example 8] Encapsulation of iron oxide nanoparticles using a dextran-linoleic acid conjugate

220 mg of the dextran-linoleic acid conjugate prepared in Example 7 and 25 ml of PBS were mixed and sonicated for 10 minutes to prepare a capsule aqueous solution. 22 mg of iron oxide nanoparticles prepared in Preparation Example 1 having an average particle diameter of 10 nm were dispersed in 0.5 ml of chloroform and then ultrasonicated for 10 minutes to prepare a nanoparticle dispersion. The nanoparticle dispersion was dripped into the dextran-linoleic acid conjugate solution (capsule aqueous solution) dropwise at a rate of 1 ml / min and mixed for 10 minutes using a homogenizer (rpm = 26,000) to form capsule particles.

FIG. 16 is a TEM photograph of the dextran-linoleic acid conjugate capsule material prepared through the above process and observed after negative staining of the capsule particles encapsulated with a plurality of iron oxide nanoparticles aggregated. FIG. TEM observation revealed that the iron oxide nanoparticles were encapsulated. The average particle size of the capsules was 155 nm as measured by dynamic light scattering (DLS).

[Example 9] Measurement of internal magnetic resonance relaxation performance of iron oxide capsules encapsulating iron oxide nanoparticles in a dextran-linoleic acid capsule material

Magnetic Resonance Imaging of Iron Oxide Capsules Encapsulated in a Dextran-Linoleic Acid Conjugate Capsule A number of iron oxide nanoparticles were encapsulated in a dextran-linoleic acid conjugate capsule. To measure the contrast agent performance, a BGA12 gradient was measured in a 4.7 T magnetic resonance imaging device (Biospec 47/40, Bruker Biospin MRI GmbH) The in vivo T2 relaxation performance was measured in the same manner as in Example 6 using a coil.

FIG. 17 shows that the liver color of the mouse was changed to black when the images before contrast injection and after contrast injection were compared, indicating that iron oxide nanoparticles encapsulated with dextran-linoleic acid conjugate can be used as hepatic contrast agent Could know.

18 is a graph showing the relaxation performance by digitizing the signal value of the image obtained in the liver magnetic resonance imaging measurement. The ΔR2 value was measured to be 35 to 45%.

[Comparative Example 3] Encapsulation of iron oxide nanoparticles using dextran

An experiment was conducted in the same manner as described in Comparative Example 1, except that dextran having a molecular weight of 10,000 was used.

19 is a TEM photograph of dextran-iron oxide nanoparticles prepared by the above method. It can be seen that the capsule nanoparticles are not encapsulated and the iron oxide nanoparticles form a large lump and that the capsule nanoparticles can not be formed by using dextrane which is a hydrophilic substance alone without using amphipathic molecules And it was found.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, Those skilled in the art will recognize that many modifications and variations are possible in light of the above teachings.

Accordingly, the spirit of the present invention should not be construed as being limited to the embodiments described, and all of the equivalents or equivalents of the claims, as well as the following claims, belong to the scope of the present invention .

Claims

Iron complexes are pyrolyzed to prepare iron oxide nanoparticles to which hydrophobic ligands are bound and then encapsulated in a capsule material of carboxymethyldextran-dodecylamine conjugate or dextran-linoleic acid conjugate to prepare iron oxide nanocapsules &Lt; / RTI >

The method according to claim 1,
a) preparing a capsule material which is a carboxymethyldextran-dodecylamine conjugate of the following formula 1 or a dextran-linoleic acid conjugate of the following formula 2;
b) pyrolyzing an iron complex in which iron is a central atom and an organic acid group including C4 to C25 is bonded with a ligand to produce iron oxide nanoparticles having a hydrophobic ligand bound thereto;
c) dissolving the capsule material in a buffer solution to prepare a capsule aqueous solution, and dispersing the iron oxide nanoparticles in a nonpolar organic solvent to prepare a nanoparticle dispersion;
d) dropping the nanoparticle dispersion in the capsule aqueous solution and stirring the mixture to prepare an iron oxide nanocapsule in which a plurality of iron oxide nanoparticles are encapsulated in a capsule material; And
e) removing the non-polar organic solvent contained in the iron oxide nanocapsule dispersion of step d) using volatilization to prepare an iron oxide nanocapsule water dispersion;
The method comprising the steps of:
(Formula 1)

(2)

3. The method of claim 2,
Wherein the capsule material is a carboxymethyldextran-dodecylamine conjugate, and the step a) is carried out by the following steps.
a1-1) mixing a solution of carboxymethyldextran and a solution of dodecylamine;
a1-2) adding EDC (1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide) and NHS (n-hydroxysuccinicimide); And
a3) dialyzing and lyophilizing;

3. The method of claim 2,
Wherein the capsule material is a dextran-linoleic acid conjugate, and the step a) is carried out by the following steps.
a2-1) mixing a dextran solution and a linoleic acid solution;
^{a2-2) DCC (n, n '} -dicyclohexylcarbodiimide) , and adding a DMAP (dimethylaminopyridine);
a3) dialyzing and lyophilizing;

The method of claim 3,
Wherein the carboxymethyldextran has an average molecular weight of 500 to 1,000,000 Da.

The method of claim 3,
The carboxymethyldextran solution is prepared by mixing 10 to 15 times the amount of carboxymethyldextran in an aqueous solution of carboxymethyldextran mixed with carboxymethyldextran: water in a weight ratio of 1: 3 to 5, ) Is added to the nanocapsule.

The method according to claim 6,
Wherein the dodecylamine solution is prepared by mixing dodecylamine: dimethyl sulfoxide (DMSO) in a weight ratio of 1: 8 to 12: 25 to 35.

8. The method of claim 7,
Wherein the carboxymethyldextran solution and the dodecylamine solution are mixed so that the dodecylamine: carboxymethyldextran is in a weight ratio of 1: 1 to 10.

8. The method of claim 7,
Wherein the EDC and NHS are added such that the concentration of dodecylamine: EDC: NHS is 1: 0.3 to 0.7: 0.1 to 0.4.

5. The method of claim 4,
Wherein the dextran has an average molecular weight of 100 to 150,000 Da.

5. The method of claim 4,
The dextran solution is a solution in which dimethylsulfoxide (DMSO) is mixed in a weight ratio of 1:50 to 80, and the linoleic acid solution is a solution in which linoleic acid: DMSO (dimethyl sulfoxide) is mixed at a weight ratio of 1: 8 to 14 By weight of the iron oxide nanocapsule.

12. The method of claim 11,
Wherein the dextran solution and the linoleic acid solution are mixed so that dextran: linoleic acid is in a weight ratio of 1: 2 to 5.

13. The method of claim 12,
Wherein said DCC and DMAP are added such that dextran: DCC: DMAP is in a weight ratio of 1: 1.5 to 2: 0.3 to 0.8.

3. The method of claim 2,
Wherein the buffer solution in step c) is a phosphate buffer solution, and the nonpolar organic solvent is chloroform.

15. The method of claim 14,
Wherein the capsule material: iron oxide nanoparticles in a weight ratio of 1: 0.05 to 0.25 is added dropwise in the step (d).

16. The method of claim 15,
And the step (d) is performed at a rate of 0.1 to 3 ml / min.

16. The method of claim 15,
Wherein the capsule aqueous solution is a solution in which a capsule material: buffer solution is mixed at a weight ratio of 1: 50 to 500, and the nanoparticle dispersion is a solution in which iron oxide nanoparticles: nonpolar organic solvent is mixed at a weight ratio of 1:10 to 100 Wherein the iron oxide nanocapsules are prepared by mixing the iron oxide nanocapsules.

17. The method of claim 16,
Wherein the stirring of step (d) is performed at 20,000 to 30,000 rpm.

Iron oxide nanoparticles in which hydrophobic ligand-bound iron oxide nanoparticles are encapsulated in a carboxymethyldextran-dodecylamine capsule material or a dextran-linoleic acid capsule material.

An inter-MRI contrast agent wherein the iron oxide nanoparticles to which the hydrophobic ligand is bound contain iron oxide nanocapsules encapsulated in a carboxymethyldextran-dodecylamine capsule material or a dextran-linoleic acid capsule material.